Genetic verification and chemical contents identification of Allamanda species (Apocynaceae)

Arunrat Chaveerach1, Sarocha Aungkapattamagul1, Tawatchai Tanee 2, Kowit Noikotr3 and Runglawan Sudmoon1* 1Department of Biology Faculty of Science, Khon Kaen University, Thailand 2Faculty of Environment and Resource Studies, Mahasarakham University, Thailand 3Department of Biology, Faculty of Science, Ramkhamhaeng University, Thailand

Abstract: Allamanda species (Apocynaceae) are popular ornamentals. Additionally, A. cathartica possesses medicinal properties whereas all other species have not been reported. This research aims to analyze genetics and chemical contents of Allamanda species existing in Thailand. The explored species are A. blanchetii, A. cathartica, A. neriifolia, A. schottii, and A. violacea. The dendrogram constructed from 16 inter-simple sequence repeat markers clearly distinguished species with genetic similarity values of 0.92-0.93 for species level and 0.50-0.76 for genus level. Diverse chemicals content in hexane extracts from A. blanchetii, A. neriifolia, A. schottii, and A. violacea were detected by gas chromatography-mass spectrometry. A high amount of squalene was found in A. blanchetii (55.81%) and A. violacea (51.09%). This content may function as a chemo preventative substance to protect people from cancer. α-Tocopherol, a form of vitamin E, was one of the predominant components found in A. violacea (26.325%), A. schottii (15.41%), and A. neriifolia (9.16%). One more substance, 9,12,15-octadecatrien-1-ol, was found to be relatively high in A. schottii (17.31%) and A. neriifolia (15.51%). Other minor and unknown compounds were also detected. The discovery of these chemicals provides an alternative and supplement for improving human well-being and pharmaceutical industries with natural resources, especially in light of the population increase.

Keywords: Allamanda (Apocynaceae); gas chromatography-mass spectrometry; genetic similarity; inter-simple sequence repeat

INTRODUCTION and allamdin. The petals give off flavonoids, namely kaempferol and quercetin. The milky sap possess Allamanda, a genus belongs to the family Apocynaceae, antibacterial and anticancer properties. Moreover, there possesses 14 species native to tropical America are claims that distilled extract of the plant curing (Middleton, 1999). The species have been popular malignant and fungal and bacterial infectious diseases. ornamentals, because of their year-round production of Medicinally, its bark, leaves, and flowers relieve vitiated large, bright yellow and purple flowers. Allamanda pitta, inflammation, constipation, ascites, and headache by species have become naturalized throughout tropical external application. In vitro studies have supported its climates, including in Thailand. Thailand is reported to usefulness against human carcinoma cells of have two native species, A. cathartica and A. schottii nasopharynx. In vivo, ethanol root extract has shown (Middleton, 1999) but various introduced species with activity against P-388 leukemia in mice diverse flower colors have long been popularly cultivated (http://archive.is/JlwpS, http://ayurvedicmedicinalplants. for ornamentation on roadsides and fences. Due to their com/plants/1276.html, http://www.asianplant.net/ fast growth in open, sunny areas with adequate rainfall, Apocynaceae/Allamanda_cathartica.htm). many species are cultivated as groundcover and for hedges and screens. There are reports showing that the active chemicals in A. cathartica are allamandin, which is a toxic iridoid lactone, Allamanda cathartica is a wild spread and well-known and cathartic, which is a defecated substance (Nayak et species, used as both ornamental and medicinal plants. al., 2006; Islam et al., 2010; http://www. The plant contains allamandin which is a toxic iridoid absoluteastronomy.com/topics/Allamanda). Daphnis nerii lactone in all parts. Plumericin, isoplumericin, plumieride, larvae died when fed by its leaves, perhaps due to and long chain esters are contained in the stem, root-bark, variations in foliar nutritional and allelochemical factors leaves, and roots. In addition, its leaves and stem yield (Hwang and Feng, 2001). Thai medicinal recipes have lactones, ursolic acid, β-amyrin, β-sitosterol, and included small amounts of its leaves, bark, and milky sap triterpenes. The root contains the antileukaemic iridoid as traditional medicines for laxative and inducing lactone and three other iridoids: allamandin, allamandicin, vomiting. When used in excess, however, it becomes a strong laxative and causes over-vomiting and sometimes *Corresponding author: e-mail: [email protected] death (Pupattanapong, 1979). Pak. J. Pharm. Sci., Vol.27, No.3, May 2014, pp.417-424 417 Genetic verification and chemical contents identification of Allamanda species (Apocynaceae)

Curiously, other species in the genus besides A. cathartica (AG)8G, (AG)8T, (CA)6AG, (CA)6GT, (CA)8CC, (CA)9A, have never been reported for their chemicals or medicinal (CA)9G, (CA) 9T, (CT)8TG, (GA)6GG, (GT)6CC, (GT)6GG, properties. Therefore, they should be characterized and CCCC(GT)6. The reaction mixtures were incubated at starting from the genetic relationships in the genus and 94°C for 3 min then amplified with the following 35 preliminary chemical identification. DNA fingerprints thermal cycles: denaturation at 94°C for 30 sec, annealing based on inter-simple sequence repeat (ISSR) analyses are at 50°C for 45 sec, extension at 72°C for 2 min, and generally used to effectively indicate genetic followed by a final extension at 72°C for 7 min on a Swift relationships, useful for identification, conservation, Maxi Thermal Cycler (Esco Micro Pte. Ltd.). sustainable uses, and plant breeding (Emel, 2010; Arslan, Amplification products were detected by using 1.2% 2011; Gradzielewska, 2011). The banding patterns reveal agarose gel electrophoresis and ethidium bromide staining genetic variation/relationship by cladogram construction. then photographed. The amplified ISSR bands were Homology, a concept critical to cladistics, can be defined scored as absent (0) and present (1) bands and these 0-1 as a similarity/distance resulting from common ancestry data were used for dendrogram constructions using (Simpson, 2006). These genetic variation markers are NTSYSpc 2.10p software (Rohlf, 1998). commonly independent of environmental factors and are more abundant than phenotypic characters. Therefore, Chemical constituent analysis they provide a clearer description of the inherent The extracts were prepared, and the chemical contents variations in the studied genome. were analyzed by GC-MS. A 25 g sample of fresh leaves was ground, mixed with 120 mL hexane solvent For chemical identification of the plant extracts gas (analytical grade), and filtered at room temperature. The chromatography-mass spectrometry (GC-MS) has been filtrate was stored at -20°C until being analyzed by the successfully used in several plant group. For examples, GC-MS. Taweechaisupapong et al. (2010) identified constituents in Boesenbergia pandurata and found geraniol and camphor The GC-MS analysis of the crude extracts was performed as the major compounds. Nadir et al. (2013) found 78 using an Agilent Technologies GC 6890 N/5973 inert MS unreported constituents from Salvia santolinifolia by GC- fused with a capillary column (30.0 m x 250 µm x 0.25 MS analysis and other methods and identified that the µm). Helium gas was used as the carrier gas at a constant species belongs to α-pinene chemotype. flow rate of 1 mL/min. The injection and mass-transferred line temperature were set at 280°C. The oven temperature This research aims to revise existing species diversity, was programmed for 70°C to 120°C at 3°C/min, then held verify genetics of the species group, and identify chemical isothermally for 2 min, and finally raised to 270°C at contents. 5°C/min. A 1 µL aliquot of the crude extract was injected in the split mode. The relative percentage of the crude MATERIALS AND METHODS constituents was expressed as the percentage using peak area normalization. Identification of the components of In 2010 the species of Allamanda were explored the crude was assigned by a comparison of the mass throughout Thailand and collected. The plants were spectra obtained with those of the reference compounds identified by the authors followed Middleton (1999) and stored in the Wiley 7N.1 library. other literatures. Genetics and chemical contents were analyzed in the observed species. Plumeria alba was RESULTS included as an outgroup for phylogenetic analysis. Species investigation and identification Genetic analysis The investigation of Allamanda in all regions of Thailand All collected samples were subjected to DNA extraction recognized five species (fig. 1) namely A. blanchetii, A. and DNA fingerprinting by ISSR method. Total genomic cathartica, A. neriifolia, A. schottii, and A. violacea DNA was extracted using the Plant Genomic DNA (vouchers A. Chaveerach 756-760, kept at Department of Extraction Kit (RBC Bioscience) then checked by using Biology, Faculty of Science, Khon Kaen University, 0.8% agarose gel electrophoresis with tris-acetate (TAE) Thailand). They are all widely distributed throughout 76 buffer and ethidium bromide staining. DNA samples were provinces of Thailand as commercial, cultivated, and diluted to a final concentration of 20 ng/µL for use as ornamental plants. DNA templates in the polymerase chain reactions (PCRs). ISSR fingerprint analysis Amplifications were carried out on each Allamanda The 16 different polymorphism primers successfully species, in 25 µL reaction mixtures consisting of GoTaq produced 1443 bands ranging in size from 300-3000 bp Green Master Mix (Promega), 0.5 µM primer, and 20 ng (fig. 2). ISSR banding patterns were used for dendrogram DNA template. Thirty two ISSR primers were screened, constructions. The dendrogram (fig. 3) shows the high the 16 primers that successfully amplified clear bands power efficiency of the ISSR data used, which clearly were as follows (5′ to 3′): (ACTG)4, (AG)7AAG, (AG)8C, distinguishes the outgroup P. alba from the ingroup. Also, 418 Pak. J. Pharm. Sci., Vol.27, No.3, May 2014, pp.417-424 Arunrat Chaveerach et al it separates each species on different branches. The as small as a single individual (Hillis, 1987). Analyses of identical species show genetic similarity levels of 0.92 (A. large sample sizes are often limited by the availability of violacea) to 0.93 (A. cathartica), while the different specimens, the expense of the analysis, and its time- species show levels of 0.50 (A. schottii and A. cathartica) consuming nature. ISSR showed the most powerful data, to 0.76 (A. blanchetii and A. violacea). suitable for Allamanda species analysis. The dendrogram clearly distinguishes between identical species with 0.92- These values are meaningful according to the 0.93 genetic similarity values and different species with phylogenetic tree, which separates each species into 0.50-0.76 genetic similarity values. different monophyletic groups. In addition, these values are in accordance with the principle provided by Weier et As there are many publications revolving chemicals in A. al. (1982). that operational taxonomic units (OUT) show cathartica, this research focused on chemical 0.85-1.00 similarity level should be recognized as the identification in the four other Allamanda species. A high same species and 0.65 similarity level should be the same amount of squalene was found in A. blanchetii and A. genus. However, ultimate interpretation of the violacea, at 55.81% and 51.09%, respectively, and a dendrogram is dependent upon the taxonomist’s minor amount was found in A. neriifolia, at 6.08%. All knowledge of the OTU. ISSR banding data using plants, animals, and humans produce squalene which is a similarity indexes supports five different species. triterpene. In the human body, it is a natural and essential component for the syntheses of cholesterol, steroid Chemicals constituent in the four studied species, hormones, and vitamin D. It may be also an anticancer identified by GC-MS substance as it possesses a chemopreventative activity The preliminary phytochemicals screening on the hexane (Smith, 2000; Owen et al., 2004). crude extract of the four Allamanda species shows the presence of different chemical compounds. The plants, α-Tocopherol, a form of vitamin E that can be absorbed their chemical compounds, retention time, and relative and accumulated in humans (Rigotti, 2007), is one of the content are displayed in table 1. The total ion predominant components of three out of four species, chromatographs (TIC) showing the peak identities of the including A. violacea, A. schottii, and A. neriifolia at compounds in the four individual species are given in fig. 26.33%, 15.41%, and 9.16%, respectively. 4. A final substance, 9,12,15-octadecatrien-1-ol, was found DISCUSSION at relatively high levels in A. neriifolia (15.51%) and A. schottii (17.31%), however, its activity has never been This exploration of Allamanda species diversity in reported. Thailand found five species, two of which are native to Thailand (A. cathartica and A. schottii), and three of In conclusion, the hexane crude extracts revealed some which are introduced species, popularly cultivated for beneficial chemicals in the four Allamanda species. The year-round flower production (A. blanchetii, A. neriifolia, discovery of such chemicals in these and other plant and A. violacea). species can provide an alternative and supplement for improving human well-being and pharmaceutical The molecular study, a subset of genomic study, used 1-2 industries with natural resources, especially in light of the individual species. These studies usually use much population increase. smaller sample size than in morphological studies, often

Table 1: Genetic similarities of Allamanda species, analyzed using DNA fingerprint data from 16 ISSR primers

A. A. A. A. A. A. A. P. alba cathartica cathartica violacea violacea blanchetii neriifolia schottii 1 2 1 2 Allamanda cathartica 1 1.00 A. cathartica 2 0.93 1.00 A. violacea 1 0.69 0.72 1.00 A. violacea 2 0.66 0.69 0.92 1.00 A. blanchetii 0.67 0.65 0.74 0.76 1.00 A. neriifolia 0.65 0.63 0.59 0.62 0.67 1.00 A. schottii 0.52 0.50 0.53 0.56 0.60 0.59 1.00 Plumeria alba 0.36 0.32 0.32 0.37 0.46 0.44 0.55 1.00

Pak. J. Pharm. Sci., Vol.27, No.3, May 2014, pp.417-424 419 Genetic verification and chemical contents identification of Allamanda species (Apocynaceae)

Table 2: Chemical constituents of studied Allamanda species

RT Relative Plant Compound Formula MW (min) content (%) Allamanda (6E,10E,14E,18E)-2,6,10,15,19,23- 55.094 C H 410 55.810 blanchetii Hexamethyltetracosa-2,6,10,14,18,22-hexaene 30 50 18.466 1,3-Ditert-butylbenzene C14H22 190 6.012 Unknown 38.179 (6E,10E,14E,18E)-2,6,10,15,19,23- A. violacea 55.100 C H 410 51.087 Hexamethyltetracosa-2,6,10,14,18,22-hexaene 30 50 (2R)-2,5,7,8-Tetramethyl-2-[(4R,8R)-(4,8,12- 62.139 C H O 430 26.325 trimethyltridecyl)]-6-chromanol 29 50 2 44.207 9,12,15-Octadecatrien-1-ol C18H32O 264 6.997 44.671 Octadecanoic acid C18H36O2 284 4.730 40.852 Hexadecanoic acid C16H32O2 256 4.089 29.659 Phenol,2,4-bis (1,1-dimethylethyl) C14H22O 206 2.217 Phenol,bis (1,1-dimethylethyl)-phenol C14H22O 206 Unknown 4.555 A. neriifolia 44.213 9,12,15-Octadecatrien-1-ol C18H32O 264 15.509 9,12,15-Octadecatrienoic acid C19H32O2 292 Ethyl (9Z,12Z)-octadeca-9,12-dienoate C20H36O2 308 Cis-11,14,17-Eicosatrienoic acid, methyl ester C21H36O2 320 (2R)-2,5,7,8-Tetramethyl-2-[(4R,8R)-(4,8,12- 62.139 C H O 430 9.156 trimethyltridecyl)]-6-chromanol 29 50 2 61.699 Icosane C20H42 282 8.653 Hentriacontane C31H64 437 56.740 Eicosane C20H24 282 8.300 Nonacosane C29H60 408 2-Methylnonadecane C20H42 282 44.665 Octadecanoic acid C18H36O2 284 7.100 68.291 24(z)-Methyl-25-homocholesterol C29H50O 414 6.709 (3S,8S,9S,10R,13R,14S,17R)-17-[(2R,5S)-5- Ethyl-6-methylheptan-2-yl]-10,13-dimethyl- C H O 414 2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H- 29 50 cyclopenta[a]phenanthren-3-ol 1,5-Dimethyl-6(1,5-dimethylhexyl)-15,16 C28H46O2 414 (6E,10E,14E,18E)-2,6,10,15,19,23- 55.088 C H 410 6.083 Hexamethyltetracosa-2,6,10,14,18,22-hexaene 30 50 40.852 Hexadecanoic acid C16H32O2 256 5.138 29.500 2,6-Bis(1,1-dimethylethyl)-4-methylphenol C15H24O 220 3.836 51.910 Hexacosane C26H54 366 2.321 48.772 Tetracosane C24H50 338 2.061 Hentriacontane C31H64 437 Tetratriacontane C34H70 479 40.699 Dibutyl phthalate C16H22O4 278 1.986 18.472 1,3-Ditert-butylbenzene C14H22 190 1.835 29.653 Phenol,2,4-bis (1,1-dimethylethyl) C14H22O 206 1.364 Phenol, bis (1,1-dimethylethyl) C14H22O 206 47.115 Tricosane C23H48 324 1.348 Hentriacontane C31H64 437 Tetratetracontane C44H90 619 2-Propenoic acid, 3-(4-methoxyphenyl)-, 2- 47.386 C H O 290 0.761 ethylhexyl ester 18 26 3 Unknown 17.841 continued…

420 Pak. J. Pharm. Sci., Vol.27, No.3, May 2014, pp.417-424 Arunrat Chaveerach et al

Table 2: Continue

RT Relative Plant Compound Formula MW (min) content (%) A. schottii 44.225 9,12,15-Octadecatrien-1-ol C18H32O 264 17.311 9,12,15-Octadecatrienoic acid, methyl ester C19H32O2 292 (3S,8S,9S,10R,13R,14S,17R)-17-[(2R,5S)-5- Ethyl-6-methylheptan-2-yl]-10,13-dimethyl- 68.309 C H O 414 15.406 2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H- 29 50 cyclopenta[a]phenanthren-3-ol 24(z)-Methyl-25-homocholesterol C29H50O 414 (22R,24S)-22,24-Dimethylcholesterol C29H50O 414 17-(5-Ethyl-6-methylheptan-2-yl)-10,13- dimethyl-2,3,4,7,8,9,11,12,14,15,16,17- C29H50O 414 dodecahydro-1H-cyclopenta[a]phenanthren-3-ol 1,5-Dimethyl-6(1,5-dimethylhexyl)-15,16 C28H46O2 414 (2R)-2,5,7,8-Tetramethyl-2-[(4R,8R)-(4,8,12- 62.139 C H O 430 9.131 trimethyltridecyl)]-6-chromanol 29 50 2 50.376 Pentacosane C25H52 352 6.205 Triacontane C30H62 422 Docosane C22H46 310 Octacosane C28H58 394 Nonacosane C29H60 408 Hentriacontane C31H64 437 Hexacosane C26H54 366 Tetracosane C24H50 338 Hexatriacontane C36H74 507 Icosane C20H42 282 Carbonmonoxide; N, 1-diphenyl-1-pyridin-2- C H FeN O 398 ylmethanimine; iron 21 14 2 3

Tricosane C23H48 324 40.846 Hexadecanoic acid C16H32O2 256 5.762 48.778 Tetracosane C24H50 338 4.518 Docosane C22H46 310 Tetratriacontane C34H70 479 Octacosane C28H58 394 Henicosane C21H44 296 Tricosane C23H48 324 Carbonmonoxide; N, 1-diphenyl-1-pyridin-2- C H FeN O 398 ylmethanimine; iron 21 14 2 3 Pentacosane C25H52 352 Icosane C20H42 282 35.059 Henicosane C21H44 296 4.341 Heptacosane C27H56 380 51.910 Hexacosane C26H54 366 3.939 Icosane C20H42 282 39.777 Tetratriacontane C34H70 479 3.753 Heptacosane C27H56 380 29.030 Docosane C22H46 310 3.663 47.110 Tricosane C23H48 324 3.026 Octacosane C28H58 394 Icosane C20H42 282 Docosane C22H46 310 continued…

Pak. J. Pharm. Sci., Vol.27, No.3, May 2014, pp.417-424 421 Genetic verification and chemical contents identification of Allamanda species (Apocynaceae)

Table 2: Continue

Relative Plant RT (min) Compound Formula MW content (%) Henicosane C21H44 296 Pentacosane C25H52 352 53.396 Heptacosane C27H56 380 2.911 Icosane C20H42 282 Unknown 20.035

Fig. 2: Examples of ISSR banding patterns. a from primer (GT)6CC; b from primer (ACTG)4

Fig. 1: Observed Allamanda species throughout Thailand. Fig. 3: The dendrogram constructed from ISSR bands A) A. cathartica; B) A. violacea; C) A. blanchetii; D) A. from 16 primers of five Allamanda species by NTSYSpc neriifolia; E) A. schottii 2.1p software

422 Pak. J. Pharm. Sci., Vol.27, No.3, May 2014, pp.417-424 Arunrat Chaveerach et al

Fig. 4: GC-MS chromatogram of hexane crude extracts from leaves of the four Allamanda species. A) A. blanchetii; B) A. violacea; C) A. neriifolia; D) A. schottii

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